Nanoscale fluidic devices include pores and/or channels formed in selected substrates. A solid-state (non-biological) nanopore may be fabricated through TEM (transmission electron microscope) drilling through a selected substrate. Solid-state nanopores can be used to analyze biological proteins. Nanofluidic channels may be fabricated by serial electron beam lithography in order to reach the desired dimensions. Channels can also be fabricated using photolithography, nanoimprint lithography and nanotransfer lithography.
Nanopores have been used as sensors for molecules such as DNA. A small passage may be arranged to separate two electrolyte-filled reservoirs, at least one of which contains target molecules. The target molecules can be drawn through the passage and their presence detected as a drop in current through the passage. The pore functions as an electrical resistor wherein the resistance scales as length over cross-sectional area. Changes in the pore cross-sectional area may occur when floppy and somewhat coiled single stranded DNA hybridizes with its complementary strand. Double stranded DNA can be fairly rigid and rod-like. The pore diameter accordingly decreases substantially resulting in a physical blockage of the ion current through the pore. The change in current can be detected.
Solid-state nanopores have proven to be extremely suitable as single molecule sensors, and therefore have significant application to a wide range of fields, including drug discovery. Synthetic nanopores, which are more stable and tunable compared with their biological counterparts, comprise a nanoscale channel or pore (<100 nm) within a thin membrane (e.g. silicon nitride) that separates two aqueous reservoirs containing the electrolyte and analyte(s). By applying an electric potential via electrodes across the membrane, charged molecules are driven (“translocated”) through the pore, allowing the nanopore to be used as a single-molecule detector. The detection principle is based on monitoring the ionic current passing through the nanopore as the electric potential is applied across the membrane. When the nanopore is of molecular dimensions, passage of molecules (e.g., proteins or DNA) causes interruptions of the “open” current level, leading to a “translocation event” signal. An electrical fingerprint is created, which can provide information such as size and shape of the molecule. Nanopores can be embedded in a nanofluidic sensor device and used in drug discovery. A schematic representation of a nanopore device containing electrolyte is shown in US 2013/0264219 A1, which is incorporated by reference herein. FIG. 1 shows measurement of open pore current (top) and currents during two translocation events (middle and bottom), with current traces on left and graphical depictions of nanopore and molecules on the right. Target molecules 20 in an electrolyte solution (e.g. KCl) are shown outside a nanopore formed in a membrane 22. A voltage source 24 and an ammeter 26 are electrically connected between electrodes 28, 29 located on opposite sides of the nanopore. In the middle translocation event shown in FIG. 1, a target molecule 20 (or untargeted molecule) passes through the nanopore without binding to a receptor. In the bottom translocation event, the molecule 20 has bound to a receptor 21, producing a current trace that differs from the middle translocation event.
It is often desirable to functionalize the nanopore inner surface with specific molecules. The inner surface of the nanopore may include a functional layer, which is a coating to functionalize the nanopore, (i.e. a coating of the nanopore chosen with a specific purpose in mind). The functional layer can be chosen or configured to interact with predetermined molecules during translocation . Typically, the functionalization layer is attached directly to the nanopore surface as a step in the nanodevice fabrication process. This functionalization of the nanopore permits single molecule sensing. The translocation of a single molecule results in a change of current through the pore. This translocation data can reveal properties of molecules going through the pore on a single molecule level. Indirect measurement techniques, like binding events inside the pore, offer a promising way to determine very specific properties of single molecules. However, incorporating only one single-binding site into a solid-state nanopore is a challenge. There are previous solutions to achieve stochastic sensing with a single-receptor modified solid-state nanopore, including use of a mixed-monolayer assembly on a gold-coated nanopore where the number of receptors inside the pore was controlled by co-adsorption of two different surfactants with different terminal functional groups (one reactive, the other inert).
Graphene is one of several crystalline forms of carbon, alongside diamond, graphite, carbon nanotubes and fullerenes. In this material, carbon atoms are arranged in a regular hexagonal pattern. Graphene can be described as a one-atom thick layer of the layered mineral graphite. High-quality graphene is very strong, light, nearly transparent, and an excellent conductor of heat and electricity. Its interaction with other materials and with light, and its inherently two-dimensional nature, produce properties unique to graphene. Graphene can also be used to create graphene nanopores, that is, nanoscale channels or pores within graphene sheets. This is most commonly done by bombarding a graphene monolayer with a focused ion beam. Nanopores can also be formed in stacks of graphene and solid state membranes. Using the STEM (scanning transmission electron miscroscopy) mode of a TEM, it has recently become possible to preserve the graphene lattice up to the edges of the nanopore and thus preserve the electrical conductivity. Importantly, graphene can be modified with functional groups (molecules chosen with a specific purpose in mind) by both covalent and non-covalent functionalization to interact with predetermined molecules during translocation. For example, graphene can be functionalized by the covalent bond formation between free radicals or dienophiles and C═C bonds of pristine graphene or non-covalently by pi-pi (π-π) stacking. The hydrophobicity of graphene for use as nanopores for DNA translocation can be tailored. Graphene oxide (GO) can be characterized as a single graphitic monolayer with randomly distributed aromatic regions and oxygenated aliphatic regions containing hydroxyl, expoxy, carbonyl and carboxyl functional groups, which can be functionalized using various chemistries known to the art.